Device for putting material into cell

ABSTRACT

The present invention relates to a device for putting material into a cell to be modified. More particularly, the present invention is directed to a device formed within one solid material and comprises, a first passage on which the cell passes; a second passage on which the material passes and connected to the first passage at a position randomly selected between both ends of the first passage; and an apparatus which applies pressure difference or electric potential difference on the first passage and the second passage, and is also directed to a process for making the device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/980,682, filed Dec. 28, 2015, which claims priority to Korean PatentApplication Nos. 10-2014-0191302, filed Dec. 28, 2014, and10-2015-0178130, filed Dec. 14, 2015, the disclosures of each of whichare incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a device for putting material into acell to be modified. More particularly, the present invention isdirected to a device formed within one solid material and comprises, afirst passage on which the cell passes; a second passage on which thematerial passes and connected to the first passage at a positionrandomly selected between both ends of the first passage; and anapparatus which applies pressure difference or electric potentialdifference on the first passage and the second passage, and is alsodirected to a process for making the device.

BACKGROUND OF THE INVENTION

Recently, various researches for new biomedical technology have beenactively progressed by means of the fusion of biotechnology, electronictechnology and nanotechnology which have been also remarkably developedlately.

Numerous attempts have been made to use patient's cells manipulated invitro for medical treatment. Various R&D projects for developing newdrug for next generation and verifying the drug target by manipulatinghuman cell have been progressed.

The cell manipulation technologies have focused on the development ofuseful cellular therapeutics. Particularly, various exertions for celltherapy which utilizes the IPS (Induced Pluripotent Stem) cells inducedby Yamanaka factor have been tried.

Yamanaka factors refer to four genes, Oct3/4, Sox2, cMyc and K1f4. Theinsertion of the four genes into the chromosome of a cell by using thevector originated from virus, can transform the somatic cell whichalready finished differentiation into pluripotent stem cell which candifferentiate into various somatic cells. The IPS cell has beenevaluated as an innovative technology which can overcome the ethicsproblem and productivity problem of the embryonic stem cell, and canalso obviate the limitations in differentiation capability of the adultstem cell.

However, the IPS cell cause a safety problem that the vector derivedfrom the virus inserted into a live-cell together with the Yamanakafactors. Also, in case of transplantation of the cell or tissuedifferentiated from the IPS cells which contains the vector derived fromthe virus into the human body, there may be another problem that tumorrisk is increased.

Therefore, new technologies for injecting various materials, such as,DNA, RNA, polypeptide, or nano-particle, directly into a cell withoutusing delivery vehicle, have been required in order to develop newcellular therapeutics which can avoid the above risk caused by using ofthe viral vector.

In the conventional cell manipulation technologies which do not employany delivery vehicle, the typical process is to damage the membrane ofthe cells by mechanical shear force, chemical treatment or by applyingelectric field and then to allow the material such as genes which existin extra-cellular fluids to flow into the cell through the damagedmembrane gaps, and to expect the damaged cell membrane to be recoveredby self-healing capacity of the cell.

A variety of cell transfection techniques, such as particle bombardment,micro-injection and electroporation, have been developed. Except for themicro-injection, these techniques are based on bulk stochastic processesin which cells are transfected randomly by a large number of genes orpolypeptides.

The disadvantage of the conventional bulk electroporation the mostwidely used process for transfection of cells is that the injected dosecannot be controlled.

Therefore, microfluidics-based electroporation has emerged as a newtechnology for individual cell transfection. The microfluidics-basedelectroporation offers several important advantages over the bulkelectroporation, including lower poration voltages, better transfectionefficiency and a sharp reduction in cell mortality.

In 2011, a nanochannel electroporation technology which expose a smallarea of a cell membrane positioned adjacent to a nanochannel to verylarge local electric field strength, was disclosed to the public (L.James Lee et al, “Nanochannel electroporation delivers precise amountsof biomolecules into living cells”, Nature Nanotechnology Vol. 6,November 2011, www.nature.com/naturenanotechnology published online onOct. 16, 2011).

The nanochannel electroporation device comprises two micro channelsconnected by a nanochannel. The cell to be transfected is positioned inone microchannel to lie against the nanochannel, and other microchannelis filled with the agent to be delivered. Themicrochannel-nanochannel-microchannel design enables the preciseplacement of individual cells. One or more voltage pulses lastingmilli-seconds is delivered between the two microchannels, causingtransfection. Dose control is achieved by adjusting the duration andnumber of pulses.

By the way, the nanochannel electroporation device disclosed in theabove prior art employs PDMS (polydimethylsiloxane) lid that coversmicrochannel and nanochannels made by polymeric resin through imprintingand formed over the chip substrate.

Therefore, the nanochannel electroporation chip described in the articleof Nature Nanotechnology, cannot avoid the chinks occurred between thepolydimethylsiloxane lid and the imprinted layer of microchannels andnanochannels made by polymeric resin, because the sealing between thelid and the channel layer of which mechanical properties is differentfrom each other, cannot be absolutely perfect.

Also, since the size stabilities of the polydimethysiloxane lid and themicrochannels and nanochannels made by polymeric resin, are low, thesealing between the lid and the layer of channels cannot be perfect.Therefore, the chinks may easily occur between the lid and the layer ofchannels. The chinks allows the infiltration of the solution whichcauses the contamination of nanochannel electroporation chip and alsogenerate various aberration of electric field and pressure differenceapplied for the transfer of cell between the channels and for injectionof the transfection agent into the cell.

Therefore, new technology which can put various materials quantitativelyinto individual cell and can control the amount of the material withoutsuch contamination or aberration caused by the contamination has longbeen anticipated in this technology field.

The inventor of the present application conceived the device for puttingmaterial into a cell without using delivery vehicle, formed within onesolid material without any sealing in order to exclude the possibilityof the occurrence of the chink intrinsically, and thereby, can obviatedisadvantages of the prior art, such as the contamination and aberrationcaused by the chink.

Therefore, the primary object of the present invention is to provide adevice for putting material into a cell, formed within one solid andcomprises: a first passage on which the cell passes; a second passage onwhich the material passes and connected to the first passage at aposition randomly selected between both ends of the first passage; andan apparatus which applies pressure difference or electric potentialdifference on the first passage and the second passage.

Another object of the present invention is to provide a process forforming a device for putting material into a cell within one solid byirradiation of LASER, which comprises the steps of: (i) forming a firstpassage on which the cell passes within the one solid by irradiation ofLASER; (ii) forming a second passage on which the material passes andconnected the second passage to the first passage at a position randomlyselected between both ends of the first passage within the one solid byirradiation of LASER; and (iii) installing an apparatus which appliespressure difference or electric potential difference on the firstpassage and the second passage.

DISCLOSURE OF INVENTION

The primary object of the present invention can be achieved by providinga device for putting material into a cell, formed within one solid andcomprises a first passage on which the cell passes; a second passage onwhich the material passes and connected to the first passage at aposition randomly selected between both ends of the first passage; andan apparatus which applies pressure difference or electric potentialdifference on the first passage and the second passage.

LASER means Light Amplification Stimulated Emission of Radiation and maybe classified into either pulsed or continuous beams. The femto-laser isone of the pulsed beam lasers. The major parameters of pulsed beamlasers are repetition rate, wavelength, pulse energy, and pulse width.Among them, the pulse width has well characterized pulsed beam laserssuch as nanosecond (ns, 10⁻⁹) lasers, pico second (ps, 10⁻¹²) lasers,and femto second (fs, 10⁻¹⁵) lasers. As such, femto-lasers indicatepulsed beam lasers having the pulse width of 1-999 femto second.

Femto second, which is one millionth of nanosecond, is indeed blazinglyshort period of time. The time scales of phenomena in nature are muchslower than femto seconds. So scientists could have made the fastestphenomena in nature easily analyzed by freezing in time. The firstfemto-laser had been invented to visualize the electron reaction inchemistry. Not only the femto-laser pules are ultrafast, but thelight-matter reaction in femto second regime becomes significantlydifferent due to the multi-photon phenomena.

The special features of femto-laser beams have enabled many previouslyimpossible processes. One is the heat-free ablation. Femto-laser doesn'trely on the heat to ablate matters, but it stripes away the valenceelectrons to ionize materials. So, the materials can be cut without theheat affected damages nearby.

And it was found that the multi-photon ionization by femto-laser pulsesis extremely deterministic and can be localized in nanometer scale,overcoming the diffraction limit of resolution in light interaction. Infemto second regime, it was proved that even visible light can achievethe nanometer scale ablation of matters.

In addition, femto-laser pulses can also ablate transparent materialssuch as glass. So it can be applied to diverse applications in medicaldevices. Considering that the difficulties of providing affordable hardX-ray light sources, femto-laser-based 3D nano-machining can greatlycontribute in the nanoscience and nanoengineering especially forbiomedical applications.

The micro channels and nano channels of the device of the presentinvention, on which the fluid containing cell flow, may be formed withinone solid material, such as glass, by irradiation of the femto-laser onthe one solid material.

Microfluidics which had emerged in 2000 mainly deals with the analysisand manipulation of biomedical samples based on the micro-scale fluidicchannel networks. The microfluidic chips have complex fluidic networks,where diverse biochemical surface treatments are engineered;electrochemical manipulations are performed; and pressure-driven orelectro osmotic flow is driven to circulate the chip.

Thus, there are a lot of standards which the microfluidic chip shouldmeet for the medical applications. And many of them can be ideally metwith the incorporation of glass as the material of the microfluidicchip. This is because the glass has long been used and approved in themedical fields. Specifically, glass can well satisfy the biologicalcompatibility, chemical resistance, electrical insulation, dimensionalstability, structural strength, hydrophilicity, and transparency.

Nevertheless, incorporating glass as the material of microfluidic chipshas difficulties and limitations originated from the etch-bond processsuch that the isotropic glass etching limits resolution, aspect ratio,and cross-sectional shape of the channels. In addition, the glass toglass bonding to complete the microfluidic chip fabrication is not onlydifficult and expensive, but it also radically limits to build truethree-dimensional structures especially in nanoscales. Instead, PDMSsilicon molding processes had wide spread owing to the fact that it ischeap, easy to replicate many times, and weak but simple bonding processto glass.

While the PDMS molding is great alternative for research purposes, it isstill limited to be used for the medical purposes due to the lowbio-compatibility, bad chemical resistance, dimensional instability,structural weakness, and incomplete bonding. The incomplete bonding caneasily allow the current and fluid to leak along the bonding interfaces.Considering that improving the performance of the microfluidic operationis dependent upon increasing the press or the electric fields, PDMSmolding would be incompatible to the medical grade microfluidicoperation in many cases.

When cells are manipulated in microfluidic chips, the pressure and theelectric potentials should not be limited to maximize the performanceand the operational freedom due to the structural weakness and theincomplete bonding issue. The structural strength can be overcome byadopting rigid and stable solids as the base material with acceptabletransparency such as glass, polymethylmethacrylate (PMMA), polycarbonate(PC), and so on. However, it is still required to significantly improvethe glass-etch-bond process to take the huge advantages of glass in themicrofluidic medical applications.

In the current glass-etch-bond processes, the lid glass on whichmicrofluidic channel networks are etched is bonded to the flat bottomone by applying heat or plasma in dust-free conditions. This process isgreat for mass-producing two-dimensional microscale fluidic chips.However, realizing true tree-dimensional nanoscale structures by theglass-etch-bond process is significantly limited by the isotropicetching and the requirement for the glass-to-glass direct bonding.

The laser processing is thus promising, where the bonding process can beremoved. Furthermore, research in the Univ. of Michigan, USA found thattrue three-dimensional nano-machining of glass is possible byincorporating the femtosecond laser pulses in 2004. Afterwards,femtosecond nanomachining has been developed, realizing the directprocessing of true three-dimensional nanoscale structures in a singleglass plate without bonding.

In most cases, it is much more convenient and efficient to preparemicroscale two-dimensional channels based on the conventional etch-bondprocess and to process the rest of the nanoscale and three-dimensionalstructures by the femto-laser nanomachining. It is because the materialremoval of femto-laser nanomachining is inevitably very slow andlocalized, whereas the etching process can be effective for the largearea processing.

The device of the present invention is formed within one solid material,such as, glass, thermoplastic polymers or thermosetting polymers, forexample, polycarbonate, acrylics, epoxy resin or polyimide. The solidmaterial is desirably selected from glass, thermoplastic polymers orthermosetting polymers. The transparency of the solid material employedfor the device of the present invention, is desirably higher than 5%.

The first passage of the device of the present invention, has an innertapered tube shape that inner diameter is reduced gradually from theboth ends to middle section. Therefore, the inner diameters of both endsof the first passage are larger than those of the middle section of thefirst passage. Desirably, the inner diameters of both ends of the firstpassage is 10 μm to 200 μm and the inner diameters of middle section ofthe first passage is 3 μm to 150 μm.

The device of the present invention may comprise one or more of thesecond passage on which the material to be injected flows in order toput several materials into a cell at one try. The inner diameter of thesecond passage is desirably 10 nm to 1,000 nm.

The cells contained in the first passage of the present device may movesby pressure difference or by electric potential difference between theboth ends of the first passage. The electric potential differencebetween the both ends of the first passage is desirably 10 V to 1,000 V,more desirably 15 V to 500 V, most desirably 20 V to 200 V.

Also, the electric potential difference between the first passage andthe second passage is desirably 0.5 V to 100 V, more desirably 0.8 V to50 V, most desirably 1.0 V to 10 V.

Another object of the present invention can be achieved by providing aprocess for forming a device for putting material into a cell within onesolid material and comprises: the steps of: (i) forming a first passageon which the cell passes within the one solid by irradiation of LASER;(ii) forming a second passage on which the material passes and connectedthe second passage to the first passage at a position randomly selectedbetween both ends of the first passage within the one solid byirradiation of LASER; and (iii) installing an apparatus which appliespressure difference or electric potential difference between the firstpassage and the second passage.

The LASER irradiated on a solid material in the present invention forforming the microchannels and nanochannels within the one solidmaterial, may desirably be pulse laser, more desirably femto-laser ofwhich pulse width is 10⁻¹⁵ second to 10⁻¹² second.

The solid material employed for the present invention, is desirably arigid material, such as, glass, thermoplastic polymers or thermosettingpolymers, for example, polycarbonate, acrylics, epoxy resin orpolyimide. The solid material of the present invention is desirablyselected from thermoplastic polymers, thermosetting polymers or glass.The transparency of the solid material employed for the device of thepresent invention, is desirably higher than 5% for the laser beammachining.

The first passage of the device of the present invention is formed bythe irradiation of the pulse laser into the inner tapered tube shapethat inner diameter is reduced gradually from the both ends to middlesection. Therefore, the inner diameters of both ends of the firstpassage are larger than those of the middle section of the firstpassage. Desirably, the inner diameters of both ends of the firstpassage is 10 μm to 200 μm and the inner diameters of middle section ofthe first passage is 3 μm to 150 μm.

The second passages which may be comprised one or more within the deviceof the present invention, are also formed within the solid by theirradiation of the pulse laser. The inner diameter of the second passageis desirably 10 nm to 1,000 nm.

The flow of the fluid containing the cell may effectively be controlledby adjusting the pressure difference or the electric potentialdifference applied on the first passage of the device of the presentinvention during watching the movement of the cells in the first passagethrough microscope.

In addition, the amount of the material to be injected into a cell maybe controlled by adjusting the electric potential difference between thefirst passage and the second passage of the present invention. Also, theamount of the material injected into a cell may be calculated i) bymeasuring the intensity of the fluorescent conjugated on the materialinjected into a cell or ii) by the electric current measured uponinjecting the material into a cell

Hereinafter, the present invention will be described in greater detailwith reference to the following examples and Figs. However, the examplesare given only for illustration of the present invention and not to belimiting the present invention within the following examples.

BRIEF DESCRIPTIONS OF FIGS

FIGS. 1a and 1b show schematic diagrams of the device of the presentinvention. Diagram a shows the device for injecting material into a cellwhich has one material passage (the second passage), and diagram b showsthe device of the present invention which has three second passages.

FIG. 2 is a schematic diagram of the device of the present invention.

FIGS. 3a and 3b shows a three dimensional structure of the first passageand the second passage of the device of present invention for injectingmaterial into a cell (image a), and image b is an enlarged diagram forthe part which connects the first passage and the second passage.

FIG. 4 is a schematic diagram which shows the process for injectingmaterial into a cell, in Examples 2 to 5 of the present invention.

FIG. 5 is the microscopic images for the device of the present inventionfor injecting material into a cell, prepared in Example 1 of the presentinvention.

FIG. 6 is a photograph of external appearance of the device of thepresent invention for putting material into a cell.

FIGS. 7a, 7b, 7c, 7d, 7e and 7f shows the microscopic images (a-f)showing the process of injecting the red fluorescent protein into thehuman alveolar basal epithelial cell A549 in Example 2 of the presentinvention.

FIGS. 8a, 8b, 8c, 8d, 8e and 8f shows the photographs (a-f) magnifyingthe cell part in the process of injecting the red fluorescent proteininto the human umbilical cord stem cell in Example 3 of the presentinvention.

FIGS. 9a, 9b, 9c and 9d show the photographs (a-d) of fluorescencemicroscope of the proceedings of injecting RFP into the human placentalstem cell in Example 4 of the present invention.

FIGS. 10a, 10b, 10c, 10d show the images (a-d) of human alveolar basalepithelial cell A549, after the lapse of 12 hours from the injection ofplasmid DNA (cy3) in Example 5 of the present invention.

FIG. 11 shows a disassembled perspective view of the laser beam machineemployed for the processing of the device of the present invention,according to Example 1.

EXAMPLE 1: PROCESS FOR FORMING THE MICRO CHANNELS AND NANO CHANNELS OFTHE DEVICE OF THE PRESENT INVENTION WITHIN ONE SOLID GLASS

Femto-laser pulses (Pharos, 4 W, 190 fs, frequency doubled 510 nm, DPSSchirped pulse amplification laser system) had been focused on the singleglass substrate through an objective lens (from 40× to 100×, N.A. from0.5-1.3, Olympus & Zeizz), where the focus can move from the outside ofthe substrate into it. The glass substrate had been placed on the 3 axislinear nano-stage (100×100×100 μm3, ±1 nm, Mad City Labs, Inc., Madison,Wis.), by which the glass substrate had been able to be controlled inthree-dimension against the focus with nanoscale accuracies.

As the focus of the femto-laser pulses had been controlled to move fromthe glass surface into it, the glass had been removed along the pathwayof the focus. The pathways of the focus had been written as G-code toautomatically machine true three-dimensional structures directly insideof the glass substrate. Thus, no glass-to-glass bonding had beenrequired.

The entire process had been monitored by CCD camera. Although theminimum size of the femto-laser ablation of glass was found as 10 nm,the setup of the example had been designed to have feature size around200 nm, and the feature size had been able to be controlled bigger orsmaller than 200 nm by adjusting the optical parameters and components.Machining true three-dimensional structures had been possible by usingtransparent material like glass.

EXAMPLE 2: PROCESS OF PUTTING RED FLUORESCENCE PROTEIN INTO HUMANALVEOLAR BASAL EPITHELIAL CELL BY USING THE DEVICE OF THE PRESENTINVENTION

RFP (dsRed fluorescence protein, MBS5303720) had been prepared bydiluting to be 1 mg/ml (solvent: PBS, Hyclone, SH30028.02, pH 7.4). A549cells (human alveolar basal epithelial cells) had been subcultured using10% FBS DMEM (high glucose) in an incubator (humidified 5% CO², 37° C.).

The subcultured cells had been separated using TrypLE (gibco). And thesolution had been replaced with the 1 mM EDTA in D-PBS (gibco) solution.Debris had been filtered using 40 μm cell strainer (BD), and then cellshad been treated with Calcein-AM in an incubator (for 15 minutes, at 37°C.). After the solution had been replaced with 1 mM EDTA in D-PBS, A549cell suspension solution of 2×10⁶ cells/ml concentration had beenprepared using hemocytometer.

A549 cell suspension and RFP had been packaged in 1 ml syringes, andeach of them had been connected with silicon tubes to the inlets of thecell loading and the material loading channels. And the other sides ofthe cell loading and the material loading channels had been connectedwith silicon tubes to the PBS filled 1 ml syringes.

By controlling all the syringes cells had been controlled to flow intothe cell loading channels and each of them had been placed at the centerof the cell loading channel where the material injection pathways werecrossed.

Then, proper electric potentials had been applied to the both side ofthe material loading channel for 3 seconds to make the electricpotential 1.76V along the material injection pathways. The electricpotential had been measured by oscilloscope (VDS3102, Owon) andepifluorescent microscope (TE2000-U, 41-17Nikon) had been used tomonitor cells and RFP injection process.

EXAMPLE 3: PROCESS OF PUTTING RED FLUORESCENCE PROTEIN INTO HUMANUMBILICAL CORD TISSUE MESENCHYMAL STEM CELL BY USING THE DEVICE OF THEPRESENT INVENTION

RFP (dsRed fluorescence protein, MBS5303720) had been prepared bydiluting to be 1 mg/ml (solvent: PBS, Hyclone, SH30028.02, pH 7.4).UC-MSCs (Human Umbilical Cord Tissue Mesenchymal Stem Cells) had beentaken from the CHA hospital at Boondang South Korea. UC-MSCs had beensubcultured using MEM-alpha (Gibco), 10% FBS (Hyclone), 25 ng/ml FGF-4(Peprotech), 1 ug/ml Heparin (Sigma) in an incubator (humidified 5% CO²,37° C.).

The subcultured cells had been separated using TrypLE (gibco). And thesolution had been replaced with the 1 mM EDTA in D-PBS (gibco) solution.Debris had been filtered using 40 μm cell strainer (BD), and then cellshad been treated with Calcein-AM in an incubator (for 15 minutes, at 37°C.). After the solution had been replaced with 1 mM EDTA in D-PBS,UC-MSC suspension solution of 2×10⁶ cells/ml concentration had beenprepared using hemocytometer.

UC-MSC suspension and RFP had been packaged in 1 ml syringes, and eachof them had been connected with silicon tubes to the inlets of the cellloading and the material loading channels. And the other sides of thecell loading and the material loading channels had been connected withsilicon tubes to the PBS filled 1 ml syringes.

By controlling all the syringes cells had been controlled to flow intothe cell loading channels and each of them had been placed at the centerof the cell loading channel where the material injection pathways werecrossed.

Then, proper electric potentials had been applied to the both side ofthe material loading channel for 3 seconds to make the electricpotential 1.45V along the material injection pathways. The electricpotential had been measured by oscilloscope (VDS3102, Owon) andepifluorescent microscope (TE2000-U, 41-17Nikon) had been used tomonitor cells and RFP injection process.

EXAMPLE 4: PROCESS OF PUTTING RED FLUORESCENCE PROTEIN INTO HUMANPLACENTA-DERIVED MESENCHYMAL STEM CELL BY USING THE DEVICE OF THEPRESENT INVENTION

RFP (dsRed fluorescence protein, MBS5303720) had been prepared bydiluting to be 1 mg/ml (solvent: PBS, Hyclone, SH30028.02, pH 7.4).PD-MSCs (Human Placenta-derived Mesenchymal Stem Cells) had been takenfrom the CHA hospital at Boondang South Korea. PD-MSCs had beensubcultured using MEM-alpha (Gibco), 10% FBS (Hyclone), 25 ng/ml FGF-4(Peprotech), 1 ug/ml Heparin (Sigma) in an incubator (humidified 5% CO²,37° C.).

The subcultured cells had been separated using TrypLE (gibco). And thesolution had been replaced with the 1 mM EDTA in D-PBS (gibco) solution.Debris had been filtered using 40 μm cell strainer (BD), and then cellshad been treated with Calcein-AM in an incubator (for 15 minutes, at 37°C.). After the solution had been replaced with 1 mM EDTA in D-PBS,PD-MSC suspension solution of 2×10⁶ cells/ml concentration had beenprepared using hemocytometer.

PD-MSC suspension and RFP had been packaged in 1 ml syringes, and eachof them had been connected with silicon tubes to the inlets of the cellloading and the material loading channels. And the other sides of thecell loading and the material loading channels had been connected withsilicon tubes to the PBS filled 1 ml syringes.

By controlling all the syringes cells had been controlled to flow intothe cell loading channels, and each of them had been placed at thecenter of the cell loading channel where the material injection pathwayswere crossed.

Then, proper electric potentials had been applied to the both side ofthe material loading channel for 5 seconds to make the electricpotential 0.87V along the material injection pathways. The electricpotential had been measured by oscilloscope (VDS3102, Owon) andepifluorescent microscope (TE2000-U, 41-17Nikon) had been used tomonitor cells and RFP injection process.

EXAMPLE 5: PROCESS OF PUTTING PLASMID DNA (CY3) INTO HUMAN ALVEOLARBASAL EPITHELIAL CELL BY USING THE DEVICE OF THE PRESENT INVENTION

Plasmid DNA (MIR7904, Mirus) had been prepared by diluting to be 10μg/20 μl. A549 cells (human alveolar basal epithelial cells) had beensubcultured using 10% FBS DMEM (high glucose) in an incubator(humidified 5% CO², 37° C.).

The subcultured cells had been separated using TrypLE (gibco). And thesolution had been replaced with the 1 mM EDTA in D-PBS (gibco) solution.Debris had been filtered using 40 μm cell strainer (BD), and then cellshad been treated with Calcein-AM in an incubator (for 15 minutes, at 37°C.). After the solution had been replaced with 1 mM EDTA in D-PBS, A549cell suspension solution of 2×10⁶ cells/ml concentration had beenprepared using hemocytometer.

A549 cell suspension and RFP had been packaged in 1 ml syringes, andeach of them had been connected with silicon tubes to the inlets of thecell loading and the material loading channels. And the other sides ofthe cell loading and the material loading channels had been connectedwith silicon tubes to the PBS filled 1 ml syringes.

By controlling all the syringes cells had been controlled to flow intothe cell loading channels, and each of them had been placed at thecenter of the cell loading channel where the material injection pathwayswere crossed.

Then, proper electric potentials had been applied to the both side ofthe material loading channel for 2 seconds to make the electricpotential 1.0V along the material injection pathways. The electricpotential had been measured by oscilloscope (VDS3102, Owon) andepifluorescent microscope (TE2000-U, 41-17Nikon) had been used tomonitor cells and RFP injection process.

After the injection of Plasmid DNA into A549 cells, the harvested cellshad been distributed in the 96 well plate with 200 μl culture fluids.After culturing for 12 hours (humidified 5% CO², 37° C.), redfluorescence inside of the cells had been induced to confirm that theplasmid DNA had been successfully expressed.

For reference to FIG. 1, the device of the present invention has onematerial passage (the second passage, image a) or has three secondpassages (the second passage, image b) are shown.

In the FIG. 1, the cell (8) to be injected with the material, movesthrough the first passage. The electrical potential difference occursbetween the second passage (2) which injects the material and the firstpassage by the external power (20). The material (2 a) is injected tothe cell (9) by the electrical potential difference between the cell andthe second passage when the cell (8) passes the narrowed middle sectionof the first passage. The cell which has been injected with material(11) is moved to the cell draw off passage (5) one after another.

In image b of FIG. 1, the device of the present invention injecting six(6) materials by employing the six (6) second passage is represented.Using the device illustrated in image b of FIG. 1, it is possible to putsix materials into a cell at a time.

In FIG. 1, it is possible to control the amount of each material to beput into an individual cell by adjusting the electrical potentialdifference between the first passage and the each second passage.

In the FIG. 2, there are the outflow and inflow channels (4) of thesolution containing the cell, the outflow (7) and inflow (6) channels ofmaterial, the passage (1) and channel (5) which take back the cell thathas been injected with material, the first passage (1) which has a shapeof narrowed middle section, and the two second passages (2,3) which isconnected to the middle section of the first passage (1).

During the cell passes through the narrowed portion of the middlesection of the first passage (1), the cell is inserted and held on theinner wall of the middle section of the first passage. By this closecontact between the cell and the wall of the middle section of the firstpassage (1), the drop of the electrical potential difference between thefirst passage (1) and second passage (2, 3) is minimized. The cell thathas been injected with the material can easily be moved to the widenedportion of the other side of the first passage.

The second passage (2, 3) plays a role as like an injection needle. Thatis, the charge focused on the second passage (2 a, 3 a) by an externalelectrical power provides the function of boring the cell membrane (orcell wall) of the individual cell which is closely contacted to thesecond passage (2 a, 3 a), and the driving force for injecting thematerial into the cell through the pore formed by the boring.

By applying pressure difference (for example by using a pump) or byapplying electrical potential difference (for example by using externalelectrical potential with direct current or alternating current) to theinflow and outflow channels (FIG. 2, 4) of solution containing the cells(8 of FIG. 1, images a and b), the cell moves through the first passage(1). After the injection of material into the cell, the cell moves tothe other side of the first passage (5 of FIG. 1, images a and b).

The inflow and outflow channels (6, 7 of FIG. 2) for material to beinjected, intake and discharge the material by pressure difference orelectrical potential difference between the inflow channel and outflowchannel. Also, during the individual cell is closely contacting with thesecond passage in the narrowed middle section of the first passage,micro pore is generated on the cell membrane (or cell wall) and then thematerial is injected into the cell by the electric potential difference.

As represented in FIG. 3, the second passages (2,3) are connected to thenarrowed middle section of the first passage (1).

The FIG. 4 illustrates a process of injecting two kinds of materialsinto a single cell. The two materials are injected (11) into the celltrapped in the narrowed middle section of the first passage (9) throughwhich the second passages (10) are connected, and then the cell injectedwith the material is retrieved through the outflow channel connected tothe first passage.

The left microscopic image of FIG. 5 shows the inflow and outflowchannel (5) of solution containing the cell, the inflow and outflowchannels (6, 7) of material which will be injected into the cell, andthe outflow channel (4) of retrieving the cell into which the materialhas been injected.

In addition to the left image in FIG. 5, the right image of FIG. 5 showsthe first passage (1) having the narrowed middle section and connectedat the both of ends to the inflow and outflow channel (5) and thechannel (4) of retrieving the cell into which the material has beeninjected. The microscopic images of FIG. 5 also show the two secondpassage (2, 3) formed within the glass and connected to the narrowedmiddle section of the first passage.

FIG. 6 is a photograph of external appearance of the device of thepresent invention for putting material into a cell.

FIG. 7 shows the photographs of fluorescence microscope of theproceedings of injecting the red fluorescence protein (RFP) into thehuman alveolar basal epithelial cell A549 in Example 2. The aliveness ofthe human alveolar basal epithelial cell after injection with the redfluorescence protein, was confirmed by means of the test of ascertaininggreen fluorescence from the human alveolar basal epithelial cell treatedwith Calcein AM (fluorescent dye).

Image a of FIG. 7 shows that the live (green fluorescence) A549 celllocates in the middle section of the first passage. Image b of FIG. 7shows that when the electrical potential difference is applied betweenthe first passage and the second passage, RFP which has been moved alongthe second passage, starts to be injected into the A549 cell (redfluorescent). Image c of FIG. 7 shows the fact that RFP is beinginjected into the A549 cell (red fluorescent) certainly. Image d of FIG.7 shows that the A549 cell (green fluorescent) is alive after the RFPinjection. Images e and f of FIG. 7 show that RFP has been injectedsuccessfully into the A549 cell after the repeating two times the aboveprocedure.

FIG. 8 shows the photographs of fluorescence microscope of theproceedings for injecting RFP into the human umbilical cord stem cells.As described in the above explanation of FIG. 7, the aliveness of thehuman umbilical stem cell was confirmed by using the Calcein AM.

Photograph a of FIG. 8 shows that the human umbilical cord stem celllocated in the middle section of the first passage is alive by means ofthe green fluorescence (14). Photograph b of FIG. 8 shows that thesecond passage is stocked with RFP by means of the red fluorescence(15), and that injection of RFP into the human umbilical cord stem cellis prepared well by means of the green fluorescence. Photograph c ofFIG. 8 shows that the injection of RFP is starting upon the applicationof the electrical field on the passages by means of the appearance ofthe brighter red fluorescence (16). Photograph d of FIG. 8 shows thatthe umbilical cord stem cell is moving to the exit side of the firstpassage by means of the red fluorescence (17). Photograph f of FIG. 8shows that umbilical cord stem cell is totally discharged from the firstpassage by means of the red fluorescence (19).

FIG. 9 shows the photographs of fluorescence microscope of theproceedings of injecting RFP into the human placental stem cell. Asdescribed in the above explanation of FIG. 7, the aliveness of the humanumbilical stem cell was confirmed by using the Calcein AM.

FIG. 10 shows the images of human alveolar basal epithelial cell A549after the lapse of 12 hours from the injection of plasmid DNA (cy3)described in Example 5.

FIG. 11 shows a disassembled perspective view of the laser beam machineemployed for the processing of the device of the present invention,described in Example 1.

Advantageous Effects

As explained above, through the device of the present invention, variousmaterials such as protein, gene, plasmid, drug, nanoparticle can beputting into live-cell. Particularly, the amount of the material putinto a single cell can be controlled by adjusting the electricalpotential difference. The amount of the material injected into eachlive-cell can be controlled quantitatively. Therefore, it is possiblefor the present invention to be applied for a great variety of cellmanipulation and development of cellular therapeutics including IPS stemcells.

The foregoing examples 1 to 5 and the description of FIGS. 1 to 11 forthe preferred embodiments should be taken as illustrating, rather thanas limiting the present invention as defined by the claims.

As will be readily appreciated by a person skilled in the art, numerousvariations and combinations of the features set forth above can beutilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thespirit and scope of the invention, and all such variations are intendedto be included within the scope of the following claims.

What is claimed is:
 1. A process for forming a device within one solidfor putting material into a cell, comprising: (i) a step of forming afirst passage on which the cell passes within the one solid byirradiation of LASER; (ii) a step of forming a second passage on whichthe material passes and connected to the first passage at a positionrandomly selected between both ends of the first passage, within the onesolid by irradiation of LASER; and (iii) installing an apparatus whichapplies electric potential difference between the first passage and thesecond passage.
 2. The process of claim 1, wherein the device comprisesone or more of the second passage.
 3. The process of claim 1, whereindiameter of both ends of the first passage are larger than diameter ofmiddle section of the first passage.
 4. The process of claim 3, whereinthe inner diameter of both ends of the first passage is 10 μm to 200 μm.5. The process of claim 4, wherein the inner diameter of middle portionof the first passage is 3 μm to 150 μm.
 6. The process of claim 5,wherein the inner diameter of the second passage is 10 nm to 1,000 nm.7. The process of claim 1, wherein the electric potential differencebetween the first passage and the second passage is 0.5 V to 100 V. 8.The process of claim 7, wherein the electric potential differencebetween the first passage and the second passage is 0.8 V to 50 V. 9.The process of claim 8, wherein the electric potential differencebetween the first passage and the second passage is 1.0 V to 10 V. 10.The process according to claim 1, the LASER is femto second laser. 11.The process according to claim 4, the transparency of the solid ishigher than 5%.
 12. The process according to claim 11, the solid isselected from the group consisting of glass, thermoplastic polymer andthermosetting polymer.